Genetic engineering of plants, which entails the isolation and manipulation of genetic material (usually in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant or plant cells, offers considerable promise to modern agriculture and plant breeding. Beneficial traits such as increased crop food values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance as well as the production of pharmaceuticals, and other useful chemicals are all potentially achievable through genetic engineering techniques.
Once a gene has been identified and synthesized or cloned, and engineered, it is still necessary to integrate it into the genome of a plant of interest so that it is stably inherited by progeny of the transformed plant. Transient transformation leads to loss of the introduced DNA and is of little use in generating transgenic plants. Stable transformation involves the chromosomal integration of functional genetic sequences so that the integrated sequences are passed on to and present in the progeny of the transgenic plants. As referred to herein, chromosomal integration includes incorporation into plastid chromosomes. Genes incorporated into plastid chromosomes will display maternal inheritance. In order to produce transgenic corn plants, stably transformed cells must also be capable of giving rise to fertile transgenic plants. In contrast, transient transformation leads to the eventual loss of the introduced DNA and is of little use in generating transgenic plants. However, it is important in optimizing some conditions involved in stable transformation and evaluating gene expression.
Electroporation has been used to introduce foreign DNA into a number of plant species. This has almost exclusively been done using protoplasts as the DNA recipient. See M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al., Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant Cell Reports, 7, 421 (1988). However, this approach has encountered difficulties with many plant species. In general, monocot protoplasts are more difficult to generate and manipulate than are protoplasts from dicots. Monocot protoplasts can be isolated and kept viable but rarely reform normal cell walls and divide, in contrast to dicots which in many circumstances will readily divide. Referring to the use of protoplasts in genetic engineering of cereals, a leading researcher in corn transformation stated:
Although transgenic cereals can be regenerated from protoplasts in rice, and one has reason to hope that this will also be possible from other cereals, it would be unfortunate if gene technology with cereals has to rely on this [the use of protoplasts] tedious, unpredictable, and unreliable method.
I. Potrykus, Biotechnology, 535 (June, 1990).
To date, there is only one report of fertile transgenic plants arising from transformed maize protoplasts. G. Donn et al., Abstracts VIIth International Congress on Plant Tissue and Cell Cult., Amsterdam A2-38 (Jun. 24-29, 1990). The transformation was by polyethylene glycol (PEG) mediated DNA uptake by the protoplasts not via electroporation. While a number of reports have disclosed the introduction of foreign DNA into Zea mays protoplasts or basal leaf segments cells or tissue by electroporation, these reports have involved either the transient transformation of the target material, or the stable transformation of tissue which is non-regenerable or was found to be non-fertile.
The difficulties associated with maintaining viability and regeneration capacity of electroporated plant protoplasts may be circumvented by the electroporation of DNA into cells which maintain a major portion of the cell wall. Attempts to do this have been unsuccessful, with one exception. J. S. Lee et al., Korean J. Genetics, 11, 65 (1989) reported successful stable transformation of tobacco (a dicot) cells. These cells were not enzyme-treated prior to electroporation. Other reports have disclosed only transient expression of the introduced DNA.
Marikawa et al., Gene, 41, 121 (1986) prepared cell suspensions directly from tobacco leaves using macerozyme. The cell suspensions were shown to be transiently transformable via electroporation, although fertility and regenerability were not determined. The treatment of cells with pectinolytic enzymes prior to electroporation in a dicot was reported to yield the transient transformation of sugar beet suspension tissue by Lindsey et al., Plant Molec. Biol, 10, 43 (1987). However, in sugar beets the use of pectinase- or pectolyase-treated cells afforded significantly lower levels of transient transformation than did the use of protoplasts. Subsequent sugar beet transformation studies by this group returned to the use of protoplasts. Lindsey et al., Plant Cell Reports, 8, 71 (1989).
Electroporation represents one of the few, if not only, methods for high frequency plant transformation. Therefore, a need exists for a method to adapt this technology to transform cereals, particularly maize, with heterologous DNA, so that the DNA is stably integrated in the plant genome and inherited by progeny of the transformed plants.